At the present time, immunoassays are widely used for the qualitative and quantitative analysis of compounds in biological fluids.
Among the techniques in existence, fluorimetric assays have become increasingly important.
In fact, they have a number of advantages, including the sensitivity and rapidity of the measurement, the stability and safety of the reagents labeled with fluorescent compounds, and the relatively low cost.
It is known that detection methods which use fluorescence are intrinsically very sensitive and could permit lower detection limits than those achieved by immunoassays which use radiolabeled reagents, in particular if modulatable laser light sources are used (I. Wieder, Immunofluorescence and related staining techniques, 1978, Elsevier).
Numerous fluorescent molecules usable as tracers in assays of this type have previously been described; among these, rare earth complexes, which possess valuable properties, may be mentioned in particular.
"Tracer" is understood as meaning either a luminescent molecule emitting a direct luminescence, or a luminescent molecule capable of inducing a luminescent emission, it being possible for said molecule to be coupled with one of the reagents of the assay, and the emission of a direct or induced luminescence enabling the target analyte to be detected and/or determined.
The use of particular complexes, rare earth cryptares, is described for example in European patent applications 0 321 353, 0 180 492 and 0 232 348 or international patent application WO 90/04791.
These rare earth cryptates have the advantage of being very stable in a saline protein medium, this property being particularly important in the case of homogeneous immunoassays.
The sensitivity of the measurement is nevertheless limited by different interference parameters, among which the following may be mentioned:
the spectroscopic properties of the medium, and in particular its intrinsic fluorescence, which is due especially to the interference emissions of the molecules present in the measuring medium and capable of being excited and of emitting at wavelengths close to those of the fluorescent tracer and/or with strong intensities; its absorption, which results in a loss of exciting light; and its light diffusion properties when the measuring medium is not clear; PA1 the quenching of the emitted fluorescence by inhibitors present in the medium; and PA1 the composition of the equipment, and especially the interference reflections caused by the equipment. PA1 1) adding, to said medium containing the target analyte, a first reagent made up of at least one receptor for said analyte, coupled with a luminescent donor compound, PA1 2) adding a second reagent made up of one or more other receptors for said analyte, said second reagent being coupled with a luminescent acceptor compound, PA1 3) incubating said medium after the addition of each reagent or after the addition of both reagents, PA1 4) exciting the resulting medium at the excitation wavelength of the luminescent donor compound, and PA1 5) measuring the signal of the luminescent donor compound at a wavelength .lambda..sub.1, this measurement serving as a reference, and the signal resulting from the energy transfer at a different wavelength .lambda..sub.2. PA1 1) adding, to said medium containing the target analyte, a first reagent made up of at least one receptor for said analyte, coupled with a luminescent donor compound, PA1 2) adding a second reagent made up of the analyte coupled with a luminescent acceptor compound, PA1 3) incubating said medium after the addition of each reagent or after the addition of both reagents, PA1 4) exciting the resulting medium at the excitation wavelength of the luminescent donor compound, and PA1 5) measuring the signal of the luminescent donor compound at a wavelength .lambda..sub.1, this measurement serving as a reference, and the signal resulting from the energy transfer at a different wavelength .lambda..sub.2. PA1 1) adding, to said medium containing the target analyte, a first reagent made up of at least one receptor for said analyte, coupled with a luminescent acceptor compound, PA1 2) adding a second reagent made up of the analyte coupled with a luminescent donor compound, PA1 3) incubating said medium after the addition of each reagent or after the addition of both reagents, PA1 4) exciting the resulting medium at the excitation wavelength of the luminescent donor compound, and PA1 5) measuring the signal of the luminescent donor compound at a wavelength .lambda..sub.1, this measurement serving as a reference, and the signal resulting from the energy transfer at a different wavelength .lambda..sub.2. PA1 1) adding to said medium a first reagent made up of a receptor for said analyte, PA1 2) adding a second reagent selected from the analyte or at least one of its receptors, one of the two reagents being coupled with a luminescent tracer compound and the other reagent containing a heavy atom or moleties containing a heavy atom, as well as a luminescent compound serving as an internal reference, PA1 3) incubating the resulting medium either after the addition of each reagent or after the addition of both reagents, PA1 4) exciting the resulting medium, and PA1 5) measuring on the one hand the signal emitted by the luminescent tracer compound, said signal being modulated by the heavy atom effect at a wavelength .lambda..sub.2, and on the other hand the signal emitted by the reference compound at a wavelength .lambda..sub.1. PA1 "analyte" defines any substance or group of analogous substances to be detected and/or determined; PA1 "receptor" defines any substance capable of binding specifically to a site on said analyte; PA1 "heavy atom" defines an atom of high atomic number whose presence in the proximity of a fluorescent molecule is capable of inducing an increase in the spin-orbit coupling of the latter; examples which may be mentioned of appropriate heavy atoms are especially halogen atoms, mercury, thallium, lead and silver; PA1 "moiety containing at least one heavy atom" defines any chemical substance which naturally contains at least one heavy atom or to which at least one heavy atom can be bound.
Together these interferences considerably affect the sensitivity and reproducibility of the measurement.
Some of these problems have already been solved by a variety of techniques.
In particular, the time-resolved methods of measuring fluorescence enable the problem of interference emissions (background) to be partially overcome. The principle of these methods consists in measuring the fluorescence emitted by a tracer molecule having a relatively long emission lifetime, the measurement being delayed in time beyond the emission lifetime of the other molecules present.
It is necessary in this case to use fluorescent tracer molecules with a relatively long lifetime, such as rare earth chelates and cryptates.
Nevertheless, the problem of the limitations due to the spectroscopic properties of the medium, and in particular to its absorption, has not been solved satisfactorily.
In fact, none of the proposed techniques for avoiding the filtering effect of the medium makes it possible to carry out the measurement easily and inexpensively and at the same time to obtain a high sensitivity and a very good reproducibility in the measurement.
In particular, the solution which consists in greatly diluting the sample detracts from the sensitivity of the detection.
Furthermore, the use of a double exciting beam system involves the use of expensive equipment and special measuring cuvettes which are difficult to standardize. Moreover, systematic measurement of the absorption of the medium prior to measurement of the fluorescence of the sample complicates the assay method.